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Tangential Flow Filtration of Haptoglobin

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Tangential Flow Filtration of Haptoglobin Received: 10 January 2020 Revised: 7 April 2020 Accepted: 21 April 2020 DOI: 10.1002/btpr.3010 RESEARCH ARTICLE Tangential flow filtration of haptoglobin Ivan S. Pires | Andre F. Palmer William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State Abstract University, Columbus, Ohio Haptoglobin (Hp) is a plasma glycoprotein that scavenges cell-free hemoglobin (Hb). Correspondence Hp has various potential therapeutic applications, but it has been mainly studied for *Andre F. Palmer, William G. Lowrie treatment of acute hemolytic conditions that can arise from situations such as mas- Department of Chemical and Biomolecular Engineering, The Ohio State University, sive blood transfusion, infusion of stored red blood cells, severe burns, trauma, sepsis, 151 West Woodruff Ave, Columbus, OH radiation injury, and others. Therefore, Hp may also be beneficial during chronic 43210. Email: [email protected] hemolytic disease states such as hereditary spherocytosis, nocturnal hemoglobinuria, sickle-cell anemia, and malaria. Various methods have been developed to purify Hp Funding information National Cancer Institute, Grant/Award from plasma or plasma fractions. However, none of these methods have exploited Number: P30 CA016058; National Heart, the large molecular weight (MW) range distribution of Hp polymers to easily isolate Lung, and Blood Institute, Grant/Award Numbers: R01HL126945, R01HL138116, Hp from other plasma proteins. The present study used tangential flow filtration R56HL123015; National Institute of (TFF) to isolate polymeric Hp from plasma proteins using human Fraction IV (FIV) as Biomedical Imaging and Bioengineering, Grant/ Award Number: R01EB021926; NIH Office of the starting material. After removal of insoluble material from a suspension of FIV the Director, Grant/Award Number: S10 paste, the protein mixture was clarified on a 0.2 μm hollow fiber (HF) TFF filter. The OD018056; Ohio State University Comprehensive Cancer Center - Arthur clarified protein solution was then bracketed based on protein MW using HF filters G. James Cancer Hospital and Richard J. Solove with MW cut-offs (MWCOs) of 750, 500, and 100 kDa. Using untreated FIV, the Hp Research Institute, Grant/Award Number: Pelotonia Fellowship Program; The Ohio State purity of the main bracket was ~75% with a total Hb binding capacity (HbBC) yield of University Office of Undergraduate 1.2 g starting from 500 g of FIV paste. However, pretreatment of FIV with fumed silica Research & Creative Inquiry, Grant/Award Number: Advanced Summer Research to remove lipoproteins increased Hp purity to >95% with a HbBC yield of 1.7 g per Fellowship 500 g of FIV. Taken together this study provides a novel and scalable method to purify Hp from plasma or plasma fractions. 1 | INTRODUCTION Furthermore, Hp binding to Hb prevents heme release from Hb, and lowers Hb-induced oxidative damage and inflammation.10,12 For these Haptoglobin (Hp) is an α-2 glycoprotein mainly responsible for reasons, Hp is used as a therapeutic in Japan and is being researched scavenging cell-free hemoglobin (Hb).1,2 Although found in most for treatment of various states of hemolysis.8,9,13,14 However, clinical bodily fluids of mammals, it is present in plasma at concentrations nor- translation of these treatments is dependent on the large-scale purifi- mally ranging from 0.5–3 mg/ml.3-5 After binding to cell-free Hb in cation of Hp from human plasma and plasma fractions. Thus, new Hp plasma, the Hb-Hp protein complex is scavenged by CD163+ macro- purification strategies are warranted for effective development of this phages and monocytes in order to clear the organism of toxic cell-free protein for treatment of hemolytic states. Hb.6Cell-free Hb toxicity is attributed to a variety of factors, including Hp is a polymorphic protein composed of αβ dimers in which the Hb extravasation into the tissue space which elicits oxidative tissue β polypeptide chain is coded by the same gene, while the α chain can injury, nitric oxide (NO) scavenging, and free heme release.7 These be coded by either the Hp1 or Hp2 codominant allele.15 These genet- factors can lead to acute and chronic vascular disease, inflammation, ics give rise to three main Hp phenotypes in humans: Hp1-1, Hp2-1, thrombosis, and renal damage.7,8 When bound to Hp, the large size of and Hp2-2.16,17 The α and β chains are bound through disulfide bonds the Hb-Hp complex prevents Hb extravasation into the tissue space, with the β, α-1 and α-2 chains having a molecular weight (MW) of thus reducing nitric oxide scavenging and vasoconstriction.8-11 approximately 36, 9 and 18 kDa, respectively.18 The difference in the Biotechnol Progress. 2020;e3010. wileyonlinelibrary.com/journal/btpr © 2020 American Institute of Chemical Engineers 1of14 https://doi.org/10.1002/btpr.3010 2of14 PIRES AND PALMER structure of the α chains results in the formation of the tetrameric anion-exchange chromatography, or recombinant Hp expression.20,33-39 Hp1-1 species (two disulfide-bonded αβ heterodimers disulfide linked 20,33-39 Chromatography can have low yields and is limited by the pro- through the α-1 chains) with a MW of approximately 89 kDa in Hp1 tein binding capacity of the column. Furthermore, affinity chromatogra- homozygotes and higher-order Hp polymers in heterozygotes (Hp2-1) phy may require the use of harsh dissociating conditions to release Hp or Hp2 homozygotes (Hp2-2).18 Heterozygotes produce Hp2-1, a lin- from the chromatography matrix. Finally, recombinant Hp requires ear Hp polymer with an average MW of about 200 kDa, which can go coexpression of proteolytic enzymes, which can have different glyco- up to about 500 kDa.19 Finally, Hp2 homozygotes produce Hp2-2 sylation patterns than plasma-derived Hp, and has not yet been shown which is a cyclic polymer form of Hp with average MW of about to easily scale-up.20 400 kDa ranging from 200 to 900 kDa.19,20 All types of Hp bind Hb This work describes a Hp production method facilitated via tan- −12 via a practically irreversible reaction, with a Kd ranging from 10 to gential flow filtration (TFF) that provides an easy, scalable, and eco- 10−15 M.21,22 Moreover, previous studies have shown that there are nomically efficient method for producing Hp from plasma fractions. no significant differences in Hp phenotype with respect to its role in The plasma fraction used in this study consisted of human Cohn Frac- Hb scavenging.17 The noncovalent linkage occurs primarily between tion IV obtained via the modified Cohn process of Kistler and the β chain of Hp to the β globin of Hb at a theoretical molar stoichi- Nitschmann.40 This plasma fraction is known to contain large MW Hp ometry of 1:1 for each αβ dimer.12 Since the MW of Hp differs (Hp2-2 and Hp2-1) from pooled plasma. Low MW Hp (small Hp2-1 between phenotypes, the mass stoichiometry is not consistent, with polymers and Hp1-1) are primarily found in Cohn Fraction V.41 There- about a 1.3:1 mass binding ratio for Hp1-1:Hb and about a 1.6:1 mass fore, the wide MW distribution of Hp polymers in Cohn Fraction IV binding ratio for Hp2-2:Hb.12 facilitates a size-based separation of Hp from other serum proteins Another related Hb binding species is Hp related protein (Hpr).5 which are primarily less than 100 kDa.42 Hpr is composed of smaller α and β chains compared to those in In the present study, two methods were developed to isolate Hp1-1, and is predominantly found as single αβ dimers, but has been Hp. The first did not employ fumed silica (FS), a product known to shown to form polymers.3,23 With >90% sequence identity to the bind to lipoproteins, while the second used FS to remove lipoproteins Hp1 gene, Hpr binds to Hb with high affinity.5 Unlike Hp, the α chains and facilitate separation via TFF. This manuscript focuses on the Hp in Hpr do not covalently bind to other α chains through disulfide purification process using FS as it yielded a higher purity Hp product. linkages to create αβ polymers, thus the Hpr polymers are thought to Yet, given potential benefits derived from retaining lipoproteins in the connect via noncovalent interactions.5,24 The physiological role of Hpr Hp product, a description of the first method and its results can be differs from normal Hp, as Hpr does not bind to the CD163 receptor found in the Supplementary Information. and does not have increased expression during states of hemolysis.5 Instead, Hpr forms a complex with high-density lipoproteins that in humans confers immunity to the trypanosoma parasite.3,5 Further 2 | MATERIALS AND METHODS information on the structure and function of Hp and Hpr can be found in various review articles.5,6,8,14,25-28 2.1 | Materials Given its function as a Hb scavenger, Hp can be used as a thera- peutic during conditions characterized by states of hemolysis Sodium phosphate dibasic, sodium phosphate monobasic, sodium (e.g., hemolytic anemia, massive blood transfusion, etc.). In these chloride, and FS (S5130) were purchased from Sigma Aldrich states, red blood cell (RBC) rupture releases cell-free Hb that can (St. Louis, MO), while 0.2 μm Millex-GP PES syringe filters were pur- scavenge NO, leading to vasoconstriction as well as formation of free chased from Merck Millipore (Bellerica, MA). A KrosFlo® Research II radicals and reactive oxygen species that can lead to oxidative damage TFF system and hollow fiber (HF) filter modules were obtained from of surrounding tissues. In Japan, clinical use of Hp has shown positive Repligen (Waltham, MA).
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